The temperature-sensitive nonsense-suppressor system is one of the most powerful tools for the study of cell physiology and regulation. I have isolated a number of high-efficiency temperature-sensitive suppressor strains of EScherichia coli K12 which promise to extend the applications of the system greatly. The strains were derived from my suts1 suppressor strain. The high suppressor efficiency of these strains results from mutations in any of three genes (sueA, sueB, sueC). I propose to characterize the sue mutations both genetically and functionally. This work is essential before the strains are adopted for general use. I also plan to investigate the effects of these mutations on other nonsense suppressors. In addition to these studies, I propose to establish the utility of these high-efficiency suppressor strains by using them to investigate the regulation of the genes which encode the subunits of RNA polymerase. The strains will be used to isolate rpoB and rpoC amber mutants. I plan to use the rpo amber mutant ts suppressor strains (along with the rpoB amber mutant strains we have already constructed), to titrate the level of the polymerase subunits and whole polymerase against the relative expression of the rpo operon genes. I also plan to use these strains to select mutants with altered regulation of the rpo genes. I plan to collaborate in these studies with P. Dennis, who will use the mutant strains to titrate the level of polymerase against the relative transcriptional activities of the rpo operons.